Early marker of proteinuria in patients treated with an anti-VEGF treatment

ABSTRACT

This document provides methods and materials related to determining whether or not a human receiving a therapy (e.g., an anti-VEGF therapy such as a bevacizumab therapy) has developed or is at risk for developing proteinuria. For example, methods and materials for detecting urinary podocytes to determine whether or not a human receiving anti-VEGF therapy has or is at risk for developing proteinuria or kidney injury are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/935,784, filed Nov. 9, 2015 (now U.S. Pat. No. 9,765,137), which is acontinuation of U.S. application Ser. No. 13/898,124, filed May 20, 2013(now U.S. Pat. No. 9,213,038), which is a continuation of U.S.application Ser. No. 13/518,358, filed Jun. 21, 2012 (now abandoned),which is a National Stage application under 35 U.S.C. § 371 and claimsbenefit of International Application No. PCT/US2010/061543 having anInternational Filing Date of Dec. 21, 2010, claims the benefit of U.S.Provisional Application Ser. No. 61/288,514, filed Dec. 21, 2009. Thedisclosure of the prior applications is considered part of (and isincorporated by reference in) the disclosure of this application.

BACKGROUND 1. Technical Field

This document relates to methods and materials involved in determiningwhether or not a human receiving an anti-VEGF therapy has podocyturia(e.g., urinary excretion of podocytes). For example, this documentprovides methods and materials for detecting the presence or absence ofpodocytes in a urine sample from a human receiving an anti-VEGF therapyto determine whether or not the human has developed or is at risk fordeveloping proteinuria or kidney injury.

2. Background Information

Angiogenesis has a critical role in the growth, invasion, and metastasisof malignancies. Using angiogenesis inhibitors as an approach to cancertreatment has made significant progress in the field of cancer therapy.Agents that target the vascular endothelial growth factor (VEGF)signaling pathway are advancing in clinical development. However, a sideeffect in about 30% of cancer patients receiving anti-VEGF therapy isproteinuria (≥300 mg of total protein in a 24-hour urine sample).Proteinuria can be associated with characteristic renal pathologicchanges of glomerular endotheliosis, leading to renal toxicity. Oneexample of an anti-VEGF therapy is bevacizumab (Avastin®), a humanizedrecombinant monoclonal antibody directed against VEGF.

SUMMARY

This document provides methods and materials related to determiningwhether or not a human receiving an anti-VEGF therapy (e.g.,bevacizumab) has developed or is at risk for developing proteinuria. Forexample, this document provides methods and materials for detectingurinary podocytes to determine whether or not a human receivinganti-VEGF therapy has or is at risk for developing proteinuria or kidneyinjury as indicated by podocyturia. Identifying patients who havepodocyturia can allow such patients, who have or are at risk fordeveloping toxicity as a result of the clinical use of drugs, to betreated effectively. In addition, identifying patients who do not havepodocyturia can avoid unnecessary cessation of anti-VEGF therapy. Asdescribed herein, the presence of urinary podocytes can be used toidentify humans receiving anti-VEGF therapy as having proteinuria or asbeing at risk for developing proteinuria as indicated by podocyturia.

In general, one aspect of this document features a method for assessinga human receiving an anti-VEGF therapy for a risk of developingproteinuria or renal injury. The method comprises, or consistsessentially of, determining whether or not a urine sample from a humanreceiving anti-VEGF therapy contains urinary podocytes or evidence ofurinary podocytes (e.g., podocyte remnants), wherein the presence ofsuch podocytes indicates that the human is at risk for developingproteinuria.

In another aspect, this document features a method for assessing a humanreceiving an anti-VEGF therapy for proteinuria. The method comprises, orconsists essentially of, determining whether or not a urine sample froma human receiving anti-VEGF therapy contains urinary podocytes orevidence of urinary podocytes (e.g., podocyte remnants), wherein thepresence of such podocytes indicates that the human has proteinuria.

In another embodiment, this document describes a method for assessing ahuman receiving an anti-VEGF therapy for proteinuria, wherein thedetection of proteinuria demonstrates renal injury.

The anti-VEGF therapy can be a bevacizumab therapy, a sunitinib therapy,a sorafenib therapy, or other known or effective therapies. The therapymay be a monoclonal or polyclonal antibody therapy.

In another aspect, a human receiving an anti-epidermal growth factorreceptor (EGFR) therapy, instead of or in addition to an anti-VEGFtherapy, can be assessed for proteinuria or the risk of developingproteinuria or kidney injury as described herein with respect to ananti-VEGF therapy.

In some cases, podocytes can be detected in, for example, a urine sampleusing an antibody against a protein expressed by podocytes, such aspodocin. In some cases, the antibody can be an antibody directed againsta podocalyxin polypeptide, a nephrin polypeptide, or a synaptopodinpolypeptide. Podocytes, or their fragments, can be detected using anyappropriate method including, without limitation, cell stainingtechniques, immunocytochemistry techniques, and flow cytometry using,for example, anti-podocin antibodies.

In another aspect, this document features a method for assessing a humanwho has received an anti-VEGF therapy for a risk of developingproteinuria. The method comprises, or consists essentially of,determining whether or not a urine sample from a human receivinganti-VEGF therapy contains one or more urinary podocytes or evidence ofone or more urinary podocytes, wherein the presence of the one or moreurinary podocytes or the evidence indicates that the human is at riskfor developing proteinuria. The anti-VEGF therapy can be selected fromthe group consisting of a bevacizumab therapy, a sunitinib therapy, anda sorafenib therapy. The determining step can comprise using an antibodyto detect podocytes. The determining step can comprise using flowcytometry to detect podocytes. The antibody can be an anti-podocinantibody. The method can comprise detecting the presence of the one ormore urinary podocytes. The method can comprise classifying the mammalas being at risk of developing proteinuria based at least in part on thepresence of the one or more urinary podocytes. The method can comprisedetecting the absence of the one or more urinary podocytes. The methodcan comprise classifying the mammal as not being at risk of developingproteinuria based at least in part on the absence of the one or moreurinary podocytes. The human can be a non-pregnant human. The method cancomprise determining whether or not the urine sample contains a level ofurinary podocytes greater than about three or more urinary podocytes(e.g., greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or moreurinary podocytes) per high power microscope field (400×).

In another aspect, this document features a method for assessing a humanwho has received an anti-VEGF or anti-EGFR therapy for proteinuria or arisk of developing proteinuria. The method comprises, or consistsessentially of, determining whether or not a urine sample from a humanreceiving anti-VEGF therapy or an anti-EGFR therapy contains one or moreurinary podocytes or evidence of one or more urinary podocytes,classifying the human as having or as being at risk of developingproteinuria if the one or more urinary podocytes or the evidence arepresent in the urine sample, and classifying the human as not having oras not being at risk of developing proteinuria if the one or moreurinary podocytes and the evidence are not present in the urine sample.The human can be a human who received the anti-VEGF therapy. Theanti-VEGF therapy can be selected from the group consisting of abevacizumab therapy, a sunitinib therapy, and a sorafenib therapy. Thedetermining step can comprise using an antibody to detect podocytes. Theantibody can be an anti-podocin antibody. The determining step cancomprise using flow cytometry to detect podocytes. The method cancomprise detecting the presence of the one or more urinary podocytes.The method can comprise detecting the absence of the one or more urinarypodocytes. The human can be a non-pregnant human. The method cancomprise determining whether or not the urine sample contains a level ofurinary podocytes greater than about three or more urinary podocytes(e.g., greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or moreurinary podocytes) per high power microscope field (400×).

In another aspect, this document features a method for identifying ahuman who has received an anti-VEGF or anti-EGFR therapy as havingproteinuria or a risk of developing proteinuria. The method comprises,or consists essentially of, detecting the presence of one or morepodocytes in a urine sample of a human who received anti-VEGF therapy oran anti-EGFR therapy, and classifying the human as having or as being atrisk of developing proteinuria based at least in part on the presence.The human can be a human who received the anti-VEGF therapy. Theanti-VEGF therapy can be selected from the group consisting of abevacizumab therapy, a sunitinib therapy, and a sorafenib therapy. Thedetecting step can comprise using an antibody to detect the one or morepodocytes. The antibody can be an anti-podocin antibody. The detectingstep can comprise using flow cytometry to detect the one or morepodocytes. The human can be a non-pregnant human. The presence of theone or more podocytes in the urine sample can be detected by detectingthe presence of remnants of one or more urinary podocytes. The methodcan comprise determining whether or not the urine sample contains alevel of urinary podocytes greater than about three or more urinarypodocytes (e.g., greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or more urinary podocytes) per high power microscope field (400×).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 contains a photograph of urinary cells plated on acollagen-coated slide, cultured for 24 hours, and stained for podocinimmunoreactivity.

DETAILED DESCRIPTION

This document provides methods and materials for determining whether ornot a human who received or is receiving an anti-VEGF therapy hasdeveloped or is at risk for developing proteinuria as indicated bypodocyturia. For example, this document provides methods and materialsfor determining whether or not a human (e.g., a non-pregnant cancerpatient) treated with an anti-VEGF therapy has an elevated level ofurinary podocytes, thereby indicating that the human has developed or isat risk for developing proteinuria. As disclosed herein, the detectionof one or more podocytes or evidence of podocytes (e.g., podocyteremnants) in the urine of a human is indicative that the human developedor is at risk of developing proteinuria. The presence of proteinuria mayindicate that the human is experiencing or at risk of developing renalinjury. Severe proteinuria may be indicative of nephrotic syndrome.

The methods and materials provided herein can be used to assess any typeof human who received or is receiving an anti-VEGF therapy, an anti-EGFRtherapy, or a combination of an anti-VEGF therapy and an anti-EGFRtherapy. For example, the methods and materials provided herein can beused to assess male or female humans for proteinuria or a risk ofdeveloping proteinuria. In some cases, the methods and materialsprovided herein can be used to assess non-pregnant humans who receivedor are receiving an anti-VEGF therapy, an anti-EGFR therapy, or acombination of an anti-VEGF therapy and an anti-EGFR therapy forproteinuria or a risk of developing proteinuria. Examples of anti-VEGFtherapies include, without limitation, bevacizumab therapies, sunitinibtherapies, and sorafenib therapies. Examples of anti-EGFR therapiesinclude, without limitation, cetuximab therapies, panitumumab therapies,trastuzumab therapies, lapatinib therapies, erlotinib therapies, andgeftinib therapies.

Any appropriate method can be used to determine the level of podocytesin a human's urine. For example, cell staining techniques that includeusing antibodies that bind to podocytes or polypeptides expressed bypodocytes can be used. Examples of such antibodies include, withoutlimitation, antibodies that have the ability to bind a podocinpolypeptide (e.g., GenBank GI No. 7657615; Accession No. NP_055440.1), apodocalyxin polypeptide (e.g., GenBank GI No. 66277202, Accession No.NP_001018121.1; GenBank GI No. 33598950, Accession No. NP_005388.2;GenBank GI No. 7271815, Accession No. AAB61574.1; and GenBank GI No.7657465, Accession No. NP_056535.1), a nephrin polypeptide (e.g.,GenBank GI No. 10441644; Accession No. AAG17141.1), a synaptopodinpolypeptide (e.g., GenBank GI No. 33323347; Accession No. AAQ07403.1), aNephl polypeptide (e.g., GenBank GI No. 14572521; Accession No.AAK00529.1.), a GLEPP1 polypeptide (e.g., GenBank GI No. 885926;Accession No. AAA82892.1), a WT1 polypeptide (e.g., GenBank GI No.AAH46461.2; Accession No. 110611793), a CD2AP polypeptide (e.g., GenBankGI No. 11321634; Accession No. NP_036252.1), an actin polypeptide (e.g.,GenBank GI No. 4501881; Accession No. NP_001091.1), an actininpolypeptide (e.g., GenBank GI No. 3157976; Accession No. AAC17470.1), acadherin polypeptide (e.g., GenBank GI No. 2281027; Accession No.BAA21569.1), a catenin polypeptide (e.g., GenBank GI No. 4503131;Accession No. NP_001895.1), an integrin polypeptide (e.g., GenBank GINo. 47078292; Accession No. NP_000203.2), a vinculin polypeptide (e.g.,GenBank GI No. 340237; Accession No. AAA61283.1), a talin polypeptide(e.g., GenBank GI No. 6682361; Accession No. AAF23322.1), a paxillinpolypeptide (e.g., GenBank GI No. 704348; Accession No. AAC50104.1), ora ZO-1 polypeptide (e.g., GenBank GI No. 116875767; Accession No.NP_003248.3). In some cases, flow cytometry techniques can be used todetermine the level of podocytes present in a human urine sample. Thepodocytes detected in a urine sample can be viable podocytes, non-viablepodocytes, or a combination thereof. In some cases, non-viable podocytescan be identifiable podocyte remnants.

An antibody can be, without limitation, a polyclonal, monoclonal, human,humanized, chimeric, or single-chain antibody, or an antibody fragmenthaving binding activity, such as a Fab fragment, F(ab′) fragment, Fdfragment, fragment produced by a Fab expression library, fragmentcomprising a VL or VH domain, or epitope binding fragment of any of theabove. An antibody can be of any type (e.g., IgG, IgM, IgD, IgA or IgY),class (e.g., IgG1, IgG4, or IgA2), or subclass. In addition, an antibodycan be from any animal including, without limitation, birds and mammals.For example, an antibody can be a human, rabbit, sheep, or goatantibody. An antibody can be naturally occurring, recombinant, orsynthetic. Antibodies can be generated and purified using anyappropriate method. For example, monoclonal antibodies can be preparedusing hybridoma, recombinant, or phage display technologies, or acombination of such technologies. In some cases, antibody fragments canbe produced synthetically or recombinantly from a gene encoding apartial antibody sequence. Any antibody that can be used to detectpodocytes (e.g., anti-podocin antibodies, anti-podocalyxin antibodies,anti-nephrin antibodies, or anti-synaptopodin antibodies) can have abinding affinity for its antigen of at least 10⁴ mol⁻¹ (e.g., at least10¹, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹² mol⁻¹). For example, ananti-podocin antibody that binds to podocin polypeptides at an affinityof at least 10⁴ mol⁻¹ (e.g., at least 10¹, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, or 10¹² mol⁻¹) can be used as described herein.

The term “elevated level” as used herein with respect to the level ofurinary podocytes is any level that is above a median level of podocytespresent in urine from a random population of non-pregnant humans knownnot to have proteinuria (e.g., a random population of 5, 10, 15, 20, 30,40, 50, 100, 500, or more non-pregnant humans known not to haveproteinuria). For example, the random population of non-pregnant humanscan be a population of non-pregnant humans receiving an anti-VEGFtherapy or a population of non-pregnant humans not receiving ananti-VEGF therapy, provided that the humans do not have proteinuria. Insome cases, the random population of non-pregnant humans can be apopulation of non-pregnant humans receiving an anti-EGFR therapy or apopulation of non-pregnant humans not receiving an anti-EGFR therapy,provided that the humans do not have proteinuria. In some cases, anelevated level of urinary podocytes can be a detectable level ofpodocytes (e.g., podocin-positive cells) within a urine sample. Thepresence or absence of such a detectable level of podocytes (e.g.,podocin-positive cells) can be determined using an anti-podocinantibody.

Once the level of urinary podocytes of a human is determined, then thelevel can assessed to determine if the level is an elevated level. Thepresence of an elevated level of urinary podocytes can indicate that thehuman has proteinuria or a risk of developing proteinuria. Additionalclinical tests can be performed to confirm that a human having anelevated level of urinary podocytes has proteinuria. For example, atotal protein determination in a 24-hour urine collection test, a urinedipstick protein analysis test, and/or a protein-creatinine ratio testin a random urine sample can be performed to confirm that a human havingan elevated level of urinary podocytes has proteinuria. If the presenceof proteinuria is confirmed by an additional clinical test (e.g., atotal protein determination in a 24-hour urine collection test) for ahuman having an elevated level of urinary podocytes, then that human canbe classified as having proteinuria. If the presence of proteinuria isnot confirmed by an additional clinical test (e.g., a total proteindetermination in a 24-hour urine collection test) for a human having anelevated level of urinary podocytes, then that human can be classifiedas being at risk of developing proteinuria.

In some cases, the level of urinary podocytes present within a human canbe assessed using a cutoff value such as 0.85 urinary podocytes presentper mg creatinine. For example, once the level of urinary podocytes of ahuman is determined, the level can compared to a cutoff value (e.g.,0.85 urinary podocytes/mg creatinine). The presence of a level ofurinary podocytes greater than or equal to the cutoff value can indicatethat the human has proteinuria or a risk of developing proteinuria.Additional clinical tests can be performed to confirm that a humanhaving a level of urinary podocytes greater than or equal to the cutoffvalue has proteinuria. For example, a total protein determination in a24-hour urine collection test, a urine dipstick protein analysis test,and/or a protein-creatinine ratio test in a random urine sample can beperformed to confirm that a human having a level of urinary podocytesgreater than or equal to the cutoff value has proteinuria. If thepresence of proteinuria is confirmed by an additional clinical test(e.g., a total protein determination in a 24-hour urine collection test)for a human having a level of urinary podocytes greater than or equal tothe cutoff value, then that human can be classified as havingproteinuria. If the presence of proteinuria is not confirmed by anadditional clinical test (e.g., a total protein determination in a24-hour urine collection test) for a human having a level of urinarypodocytes greater than or equal to the cutoff value, then that human canbe classified as being at risk of developing proteinuria.

In some cases, a human treated with an anti-VEGF therapy, an anti-EGFRtherapy, or both can be classified as having proteinuria or a risk ofdeveloping proteinuria if it is determined that the podocyte level in aurine sample from the human treated with the therapy (e.g., theanti-VEGF therapy, anti-EGFR therapy, or both) is greater than thepodocyte level in a urine sample obtained from that human at an earliertime point (e.g., at a time prior to any treatments with an anti-VEGFtherapy or an anti-EGFR therapy). Additional clinical tests can beperformed to confirm that a human experiencing such an increase inurinary podocytes over time has proteinuria. For example, a totalprotein determination in a 24-hour urine collection test, a urinedipstick protein analysis test, and/or a protein-creatinine ratio testin a random urine sample can be performed to confirm that a humanexperiencing an increase in urinary podocytes over time has proteinuria.If the presence of proteinuria is confirmed by an additional clinicaltest (e.g., a total protein determination in a 24-hour urine collectiontest) for a human experiencing an increase in urinary podocytes overtime, then that human can be classified as having proteinuria. If thepresence of proteinuria is not confirmed by an additional clinical test(e.g., a total protein determination in a 24-hour urine collection test)for a human experiencing an increase in urinary podocytes over time,then that human can be classified as being at risk of developingproteinuria.

In some cases, the detection of one or more podocytes in a urine samplecan be indicative of podocyturia. For example, a non-pregnant patientreceiving an anti-VEGF therapy or anti-EGFR therapy (or both) can beclassified as having proteinuria or a risk of developing proteinuriabased at least in part on the detection of one or more podocytes in aurine sample. Additional clinical tests can be performed to confirm thata human having one or more detectable urinary podocytes has proteinuria.For example, a total protein determination in a 24-hour urine collectiontest, a urine dipstick protein analysis test, and/or aprotein-creatinine ratio test in a random urine sample can be performedto confirm that a human having one or more detectable urinary podocyteshas proteinuria. If the presence of proteinuria is confirmed by anadditional clinical test (e.g., a total protein determination in a24-hour urine collection test) for a human having one or more detectableurinary podocytes, then that human can be classified as havingproteinuria. If the presence of proteinuria is not confirmed by anadditional clinical test (e.g., a total protein determination in a24-hour urine collection test) for a human having one or more detectableurinary podocytes, then that human can be classified as being at risk ofdeveloping proteinuria.

A human treated with an anti-VEGF therapy, an anti-EGFR therapy, or bothcan be monitored using the methods and materials provided herein.Podocytes may appear in a patient's urine before any proteinuria isdetected, thus providing an early predictor of a pending adverse event.This early indication may allow health care personnel to alter apatient's anti-VEGF or anti-EGFR treatment plan or to begin treatmentfor proteinuria. For example, the level of podocytes in a urine samplecan be assessed to determine whether or not proteinuria persists inresponse to decreasing the dose of anti-VEGF therapy or anti-EGFRtherapy.

This document also provides methods and materials to assist medical orresearch professionals in determining whether or not a human treatedwith an anti-VEGF therapy, an anti-EGFR therapy, or both has or isdeveloping proteinuria. Medical professionals can be, for example,doctors, nurses, medical laboratory technologists, and pharmacists.Research professionals can be, for example, principal investigators,research technicians, postdoctoral trainees, and graduate students. Aprofessional can be assisted by (1) determining the level of urinarypodocytes in a urine sample, and (2) communicating information about thelevel to that professional. In some cases, a professional can beassisted by (1) assessing a urine sample of the presence of urinarypodocytes, and (2) communicating information about the presence ofurinary podocytes to that professional.

Any appropriate method can be used to communicate information to anotherperson (e.g., a professional). For example, information can be givendirectly or indirectly to a professional. In addition, any type ofcommunication can be used to communicate the information. For example,mail, e-mail, telephone, and face-to-face interactions can be used. Theinformation also can be communicated to a professional by making thatinformation electronically available to the professional. For example,the information can be communicated to a professional by placing theinformation on a computer database such that the professional can accessthe information. In addition, the information can be communicated to ahospital, clinic, or research facility serving as an agent for theprofessional.

This document also provides methods and materials for treating humanswith an anti-VEGF therapy such as bevacizumab, an anti-EFGR therapy, orboth under conditions that reduce the likelihood of inducingproteinuria. For example, a human needing an anti-VEGF therapy (e.g., ahuman cancer patient needing bevacizumab treatment) can be administereda standard dose of bevacizumab (e.g., 5-15 mg/kg/day) for a limitednumber of dosing cycles administered once every week, every two weeks,or every three weeks. At least once within the first 3 days oftreatment, a urine sample can be obtained and assessed for urinarypodocytes as described herein. If urinary podocytes are detected or anelevated level of urinary podocytes is detected, then the dose of theanti-VEGF therapy can be reduced by at least 30 percent (e.g., 30, 40,50, 75, 80, 90, or 100 percent) for a period of time. Alternatively,anti-VEGF therapy can be skipped for one or two dosing cycles for aperiod of time so medication is received every four weeks or every sixweeks. After this period of time, a urine sample can be obtained andassessed for urinary podocytes as described herein. If urinary podocytesare not detected or an elevated level of urinary podocytes is notdetected, then the dose of the anti-VEGF therapy can be increased (e.g.,increased to the original dose or to some degree less than the originaldose). If urinary podocytes are detected or an elevated level of urinarypodocytes is detected, then the dose or the frequency of the anti-VEGFtherapy can be reduced further for an additional period of time (e.g.,once every week, every two weeks, every three weeks, or more cycles).The assessment of urine for urinary podocytes or urinary podocyte levelsand the adjustment of anti-VEGF therapy dosage can be repeated multipletimes until a dose of anti-VEGF therapy is reached that does not induceproteinuria as determined by the presence or level of urinary podocytesdetected.

It is noted that while the methods and materials described areunderstood for patients receiving anti-VEGF therapy, anti-EGFR therapy,or both, patients receiving other therapies, such as non-steroidalanti-inflamatory drugs, bisphosphonate, trastuzumab, capecitabine,paclitaxel, or anthracycline therapy may also be assessed for havingproteinuria or being at risk for developing proteinuria as indicated bypodocyturia.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—a Case of Podocyturia in a Patient ReceivingAnti-VEGF Therapy with Sunitinib

Case Study

A 67 year old female presented with a 7.2×6.1 cm mass involving thecentral and lower portion of the right kidney. Renal function was normalwith a creatinine of 1.0 and no proteinuria. She subsequently underwenta right radical nephrectomy and retroperitoneal lymphadenectomy.Pathology confirmed Grade 2, T2, N0, M1 renal cell carcinoma, clear celltype. Sunitinib therapy was initiated, with good response.

Four months into therapy, renal function worsened with a peak creatinineof 1.5 and 467 mg of protein in 24 hours. Angiotensin receptor blockadewith Diovan was initiated. Additional side effects includedhypertension, hand and foot syndrome, and drug-induced hypothyroidism.

After seven months of therapy, her creatinine improved to 1.3 but sheremained proteinuric, with a predicted 24 hour protein of 931 mg. Atthat time, a podocyturia assay was performed as described below and thepresence of urinary podocytes was confirmed (FIG. 1).

Detection of Podocytes

A random urine sample (25-50 mL each) was centrifuged for 8 minutes at700 g at room temperature. The pellets were rinsed twice with humandiploid fibroblast (HDF) solution. Next, the pellets were resuspended inDulbecco's modified eagle's medium (DMEM) F-12 medium with 10% fetalbovine serum that was supplemented with antibiotics for the preventionof bacterial contamination. A one-milliliter aliquot was plated on acollagen-coated tissue culture slide, which was followed by overnightincubation at 37° C. in 5% CO₂. The next day, the media were removed,followed by two phosphate-buffered saline solution washes. The slide wasfixed with 1 mL of ice cold methanol for 10 minutes at −20° C. The slidewas incubated with antibodies to podocin (dilution, 1:200). After beingwashed with phosphate-buffered saline solution, a secondary fluoresceinisothiocyanate-labeled antibody was added at a dilution of 1:40 for 30minutes. The sediment was counterstained with Hoechst nuclear stain tofacilitate differentiation of whole cells from cell fragments.Coverslips were mounted with Vectashield (Vector Labs, Burlington,Calif.), and the slides were viewed with a fluorescence microscope(Leica, Germany). Nucleated, positive-staining cells were considered tobe podocytes. A renal pathologist, who was blinded to the clinicaldiagnosis and laboratory findings, evaluated each sample to determinethe number of cells that were present and the percentage of cells thatwere stained for podocin.

For the patient presented, the regimen of Sunitinib was changed, and thedose was decreased. Her kidney function, as per creatinine measurements,improved. She remains proteinuric, but her kidney function remainsstable.

A test is positive for proteinuria if the number of podocytes expressedas a ratio to the creatinine content of the respective urine sample isgreater than 0.85 podocytes/mg creatinine.

The results provided herein indicated that an elevated level of urinarypodocytes is a marker of proteinuria in humans being treated with ananti-VEGF therapy. In controls, two patients on anti-VEGF therapywithout proteinuria, podocyturia was not observed.

Example 2—Podocyturia in Patients Treated with VEGF Blocking Therapy forCancer

The following was performed to determine whether podocyturia is presentin patients who, while undergoing anti-VEGF therapy, develop proteinuria(Table 1). In addition, urinary podocyte excretion was compared amongpatients on anti-VEGF therapy with proteinuria ranging from 101 to 9720mg/d (Table 1).

TABLE 1 Type of Anti- Cancer Age/Sex VEGF GFR Proteinuria Cells/HPFCholangio 68/F B 66  420 mg/d 0 Renal Cell 60/M S/nib 39  101 mg/d 1Colorectal 55/F B 137 1 + dipstick 1 Colon 66/F B + S/nib 77  330 mg/d 0GBM 59/F B + S/nib 78  152 mg/d 1 Renal Cell 73/M B 63 2144 mg/d >3Renal Cell 67/F Sunitib 43 2112 mg/d >3 SBC 68/M B 59 6361 mg/d >3 GBM70/M S/nib 70 9720 mg/d >9 GBM: glioblastoma multiforme; SBC: smallbowel carcinoid; B: bevacizumab; S/nib: sorafenib; HPF: 400 × high powerfield.

These results demonstrate a higher degree of podocyturia in patientsundergoing anti-VEGF therapy with proteinuria in excess of 2 gr/dcompared to those treated with the same agents and proteinuria <0.5gr/d.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for treating a human having cancer,wherein said method comprises: (a) administering an original dose of ananti-VEGF therapy to said human to treat said cancer, and (b)administering a subsequent dose of said anti-VEGF therapy to said human,wherein said human, following administration of said original dose, wasidentified as having a urine sample that lacks podocytes or polypeptidesexpressed by podocytes, wherein said subsequent dose is the same amountas said original dose.
 2. The method of claim 1, wherein said anti-VEGFtherapy is selected from the group consisting of a bevacizumab therapy,a sunitinib therapy, and a sorafenib therapy.
 3. The method of claim 1,wherein said human is a non-pregnant human.
 4. The method of claim 1,wherein said administering said original dose comprises administeringbetween 5 and 15 mg of said anti-VEGF therapy per kg of body weight ofsaid human per day once every week, once every two weeks, or once everythree weeks.
 5. The method of claim 1, wherein said urine sample wasobtained from said human within at least three days of administration ofsaid original dose.